Lab 3 Serum Protein Electrophoresis Serum protein electrophoresis

Lab 3 Serum Protein Electrophoresis

Serum protein electrophoresis is an easy method of separating serum proteins based on their net charge, size, and shape. § A small amount of serum is placed on a specific medium (such as agarose) and an electrical charge is applied. § The proteins then migrate across the medium in a characteristic manner, due to the net charge and size and shape of the protein. § In routine serum protein electrophoresis, the protein will separate into five main components, identified as albumin and the globulins (alpha 1, alpha 2, beta and gamma). §

Approximately 60% of the total protein in the serum is albumin, and represents the largest peak that lies closest to the positive electrode, while the remaining fractions are composed mainly of globulins § Globulins comprise a much smaller fraction of the total serum protein but represent the primary focus of interpretation of serum protein electrophoresis. § The γ region contains the largest portion of globulins, therefore monoclonal gammopathies are most frequently encountered in the electrophoresis §

+ ve electrode -ve electrode

§ § § Albumin represents the highest peak in serum protein electrophoresis, usually seen as a single, tall band. Occasionally, two equally staining bands, referred to as bisalbuminemia or a widely staining band may be seen, which both represent normal variations. Common conditions associated with decreased albumin include malnutrition, cirrhosis and nephrotic syndrome. Dehydration, on the other hand, causes a high albumin. Absence of albumin, known as analbuminaemia, is rare

The alpha-1 fraction includes α-1 antitrypsin, transcortin, and thyroid-binding globulin. § α-2 fraction is comprised of ceruloplasmin, α-2 macroglobulin, and haptoglobin. § Both alpha-1 and 2 represent the acute phase reactants; hence, malignancy, infection or any inflammatory condition cause their elevation. §

A relative increase in α-2 fraction may be seen in nephrotic syndrome due to the relative larger size of the proteins and the inability to pass through the glomeruli. § A decrease in α-1 component may be seen in α-1 antitrypsin deficiency § A decrease in α -2 component may be seen in hemolytic anemia due to decreased haptoglobin levels. §

The beta zone consist of β-1 and β-2 but is often represented a graphically as a single band. § β-1 consists mostly of transferrin, and is increased in conditions such as iron-deficiency anemia, pregnancy and estrogen therapy. B -lipoprotein and C 3 complement are included in the β -2 component. § Ig. A has an anodal mobility and migrates in the region between the beta and gamma zones also causing a beta/gamma fusion in patients with cirrhosis, respiratory infection, skin disease, or rheumatoid arthritis (increased Ig. A). §

Immunoglobulins mainly comprise this area including Ig. G, Ig. A, Ig. M, Ig. D and Ig. E, but note that Igs may be found in the α and β zones. § If the γ zone shows an increase (or spike), the first step in interpretation is to establish if the region is narrow or wide. § If it is elevated in a single narrow "spike-like" manner it could indicate monoclonal production of a single immunoglobulin (monoclonal gammopathy), while a broad "swell-like" manner (wide) indicates polyclonal immunoglobulin production. §

Various inflammatory, autoimmune and hematologic, and non-hematologic diseases are associated with an increased gamma peak § Agammaglobulinemia and hypogammaglobulinemia syndromes such as Ig. A deficiency are associated with a decrease in this area. §

Standard SPE separates the protein into five distinct bands but, by modifying the electrophoretic parameters, proteins can be further separated into as many as 12 bands. § The modification, known as HRE, uses a higher voltage coupled with a cooling system in the electrophoretic apparatus and a more concentrated buffer. §

1 ST : Indications based on clinical findings: § Suspected multiple myeloma, Waldenström’s macroglobulinemia, primary amyloidosis or other related disorders § Unexplained bone pain or fracture § Recurrent infections § Unexplained peripheral neuropathy (not able to be attributed to another condition, e. g. type 2 diabetes, chemotherapy) § Unexplained proteinuria

2 ND : Indications based on laboratory findings § High (or low) total serum globulin or immunoglobulin § Extremely high percentage of lymphocytes § Unexplained anaemia (multiple myeloma is a recognised cause of non-iron deficiency anaemia) or other persisting cytopaenias for which there is no other explanation § Unexplained high ESR (>50) with a normal CRP § Unexplained hypercalcaemia or renal impairment § Red cell rouleaux formations noted on the peripheral blood smear § Presence of urine free light chains (Bence-Jones proteinuria)

§ The specimen most often used to determine the total protein is serum rather than plasma. § Container : Red-top tube, serum separator tube (SST) § A fasting specimen is not needed. § Interferences in some of the methods occur in the presence of lipemia; hemolysis falsely elevates the total protein result because of the release of RBC proteins into the serum.

When an abnormality is found in the total protein or albumin, an electrophoresis is usually performed. § In the standard method for serum protein electrophoresis (SPE) : 1. serum samples are applied close to the cathode end of a support medium that is saturated with an alkaline buffer (p. H 8. 6). 2. The support medium is connected to two electrodes and a current is passed through the medium to separate the proteins. 3. All major serum proteins carry a net negative charge at p. H 8. 6 and migrate toward the anode. §

Many scanning densitometers compute the area under the absorbance curve for each band the percentage of total dye that appears in each fraction. § The concentration is then calculated as a percentage of the total protein that was determined by one of the protein methods, such as the biuret procedure. § v For example: n Spike is 60% of total protein n Total protein=12 grams/deciliter (g/d. L) n Spike level=7. 2 g/d. L (60% of 12)

A reference serum control is processed with each electrophoretic run § Reference values for each fraction are as follows: 1. Albumin : 53– 65% of the total protein (3. 5– 5. 0 g/d. L) 2. α-1 : 2. 5– 5% (0. 1– 0. 3 g/d. L) 3. α-2 : 7– 13% (0. 6– 1. 0 g/d. L) 4. β : 8– 14% (0. 7– 1. 1 g/d. L) 5. γ : 12– 22% (0. 8– 1. 6 g/d. L) §

Note that : § The great advantage of electrophoresis compared with the quantitation of specific proteins is the overview it provides. § The electrophoretic pattern can give information about the relative increases and decreases within the protein population, as well as information about the homogeneity of a fraction.

§ In α 1 -antitrypsin deficiency , there is marked decrease in α 1 -fraction

§ In nephrotic syndrome, the patient loses serum albumin and low-molecular-weight proteins in the urine. Some Ig. G is also lost. § At the same time, an increase occurs in 2 -macroglobulin, -lipoprotein, complement components, and haptoglobin. § These lead to a dramatic decrease in the relative amount of albumin and a significant increase in the relative amounts of 2 -globulin fractions

§ An inflammatory pattern indicating an inflammatory condition is seen when there is a decrease in albumin and an increase in the α 1 -globulins (α 1 -acid glycoprotein, α 1 -antitrypsin), α 2 -globulins (ceruloplasmin and haptoglobin), and -globulin band (C-reactive protein). § This pattern, also called an acute-phase reactant pattern, is seen in trauma, burns, infarction, malignancy, and liver disease

§ The electrophoretic pattern of serum proteins in liver disease shows the decrease in serum albumin concentration and the increase in β-globulin. § In the pattern in cirrhosis of the liver, there is fast-moving globulin prevent resolution of the β - and γ-globulin bands. This is known as the β- γ bridge of cirrhosis

§ Probably the most significant finding from an electrophoretic pattern

A dense narrow band that is composed of a single class of immunoglobulins secreted by an abnormally expanded clone of plasma cells is known as M-protein (paraprotein, monoclonal protein or M-component). § An M protein is characterized by the presence of a sharp, well-defined band with a single heavy chain and a similar band with a kappa or lambda light chain. § Diseases that produce an increase in the γ-globulin level include Hodgkin’s disease, malignant lymphoma, chronic lymphocytic leukemia, connective tissue diseases, liver diseases, multiple myeloma, Waldenström’s macroglobulinemia, and amyloidosis §

Polyclonal Immunoglobulin disease § A polyclonal gammopathy is characterized by a broad diffuse band § Infectious, Inflammatory or various reactive processes may be associated with a broad-based peak or band in the gamma region. § This pattern suggests a polyclonal increase in immunoglobulins. Liver disease, autoimmune disease, chronic viral or bacterial infections

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